Selective enumeration and identification of mixed cultures of ...

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Kitts, Cano, & Tong, 2002). The presence of multiple and
closely related species in these products makes the
differential enumeration of probiotic and yoghurt starter
bacteria difficult due to similarity in growth requirements
and overlapping biochemical profiles of the species.
Numerous media have been proposed for selective and
differential enumeration of lactobacilli and bifidobacteria
in mixed bacterial populations, and some have been the
subject of specific reviews (Charteris, Kelly, Morelli, &
Collins, 1997; Shah, 2000; Roy, 2001; Coeuret, Dubernet,
Bernardeau, Gueguen, & Vernoux, 2003) and of comparative
performance analyses (Payne, Morris, & Beers, 1999;
Talwalkar & Kailasapathy, 2004; Masco et al., 2005; Van
de Casteele et al., 2006). In order to cover a high spectrum
of species, most media for selective enumeration of mixed
cultures have complex compositions that include antibiotics
as selective ingredients, which could impact on the
response of not only the sensitive strains but also of target
bacteria and result in inaccurate or irreproducible quantitative
results. A comparison of methods described in
literature (Talwalkar & Kailasapathy, 2004) concluded that
no reliable techniques are yet developed to accurately
enumerate L. acidophilus, L. casei and Bifidobacterium in
different commercial yoghurts. Overall, it seems rational
that the choice of selective methods should focus on the
type of food and the species, even strains, to enumerate in
each particular situation (Lourens-Hattingh & Viljoen,
2001; Sartory, 2005).
As previously stated, identification of species is another
important issue to be verified for the compliance of
fermented milk with the required product specifications
in terms of accurate species labelling and, if appropriate, to
support health claims that could be associated with added
probiotics. Phenotypic methods alone are inadequate for
identification of lactobacilli and bifidobacteria species
(Dellaglio & Felis, 2005). To achieve a rapid and reliable
identification of species, polymerase chain reaction (PCR)based
methods using species-specific primers targeting the
16S rRNA gene sequence diversity have become very
popular (Coeuret et al., 2003). In addition, cultureindependent
methods for bacterial identification based on
genetic analysis have become a valuable tool, since these
techniques have the advantage to analyze the product as a
whole. Separation of genus or species-specific PCR
products by denaturing gradient gel electrophoresis
(DGGE) has become the most commonly used technique
among the culture-independent methods for detection and
identification of lactobacilli and bifidobacteria from
fermented products (Ercolini, 2004).
The aim of this study was to develop selective plating
methodologies for enumeration and identification of mixed
cultures of S. thermophilus, L. delbrueckii subsp. bulgaricus,
L. acidophilus, L. paracasei subsp. paracasei and B. lactis in
fermented milk products based on selective antibiotic-free
media and different incubation conditions. To evaluate the
performance of selective media for complete recovery of
viable bacteria, methods were validated on the basis of
ARTICLE IN PRESS
R. Tabasco et al. / International Dairy Journal 17 (2007) 1107–1114
their precision, accuracy, reproducibility, selectivity and
specificity characteristics, in relation to culture conditions,
which were used as reference methods. Efficacy of the
selective methods was verified by identification of the
presumptive colonies using species-specific PCR. The study
is also complemented with the application of a cultureindependent
procedure based on PCR–DGGE analysis to
the rapid detection and identification of the mixed species
in fermented milk products.
2. Material and methods
2.1. Microorganisms and culture conditions
Strains used in the assay were S. thermophilus STY-31,
L. delbrueckii subsp. bulgaricus LBY-27, L. acidophilus LA-
5, L. paracasei subsp. paracasei LC-01, and B. lactis BB-12.
The strains were purified from a commercial synbiotic
product (Simbiotic Drink; Prie´gola, Madrid, Spain). To
allow the correct identification of strains, 16S rRNA gene
nucleotide sequencing was carried out from pure cultures.
The entire gene was amplified using the primers SacI-
POmod and SalI-T7-PC5 (Table 1) and the PCR conditions
described previously by Rodtong and Tannock
(1993). Additional primers used to assist in sequencing
were 16Smidfor and P3rev (Table 1). Sequencing of PCR
fragments was carried out for both strands at the DNA
Sequence Service of the Centro de Investigaciones Biolo´gicas-CSIC
(Madrid, Spain). S. thermophilus was grown in
M-17 broth (Pronadisa, Madrid, Spain) containing 2%
lactose. Lactobacillus subsp. and B. lactis were grown
under anaerobic conditions (Gas-Pack, Anaerogen; Oxoid
Ltd., Hampshire, England) in MRS broth (Pronadisa)
supplemented with 0.05% L-cysteine hydrochloride, excepting
L. paracasei subsp. paracasei that was grown
aerobically in MRS broth. Incubations were carried out for
18–24 h at 37 1C and at 30 1C for L. paracasei subsp.
paracasei.
2.2. Selective methods
Culture conditions described above were selected as
reference methods. Media were supplemented with 1.5%
bacteriological agar (Scharlab, Barcelona, Spain) and
incubation extended to 48 h for S. thermophilus and 72 h
for B. lactis and lactobacilli.
The selective conditions for the enumeration of
S. thermophilus included inoculation of appropriate dilutions
by the pour-plate technique into M-17 agar containing 1%
lactose (M17-lactose) and incubation at 45 1C for 24 h. For
enumeration of L. delbrueckii subsp. bulgaricus, appropriate
dilutions were pour-plated into MRS fermentation
broth (Pronadisa), which does not contain either glucose or
meat extract (De Man, Rogosa, & Sharpe, 1960), enriched
with 0.2% Tween 80 and supplemented with 1% fructose,
0.8% casein acid hydrolysate, 0.05% cysteine, and 1.5%
agar (MRS-fructose). Plates were incubated in anaerobic